Process for making 6-hydroxy-3-keto-{66 1,4-steroids of the pregnane and androstane series

ABSTRACT

6-hydroxy-3-keto- Delta 1,4-steroids of the pregnane or androstane series are made by fermenting a 3 Beta -hydroxy or 3 Beta -acyloxy-5,6-epoxy steroid of the pregnane or androstane series which is saturated in the A-ring with bacteria of the genus Bacillus, Mycobacterium or Arthrobacter or enzymes thereof.

United States Patent [72] Inventors Klaus Kieslich Berlin; WolfgangKoch, Darmstadt-Arheilgen, both of Germany [21] Appl. No. 12,878

[22] Filed Jan. 28, 1970 [45] Patented Nov. 30, 1971 [7 3] AssigneeSchering Aktiengesellschalt Berlin, Germany [32] Priority Jan. 27,1969

[33] Germany [54] PROCESS FOR MAKING 6-HYDROXY-3-KETO- [HA-STEROIDS OFTHE PREGNANE AND ANDROSTANE SERIES 18 Claims, No Drawings [52] US. Cl195/51 R, 260/343.2, 260/397.47, 260/239.55, 260/397.45

[51 Int. Cl ..C07c 167/00 [50] Field of Search 195/51 [56] ReferencesCited OTHER REFERENCES Lee et 211., Biochemistry, Vol. 3. pages 1267-1.271, Sept.. 1964.

Primary Examiner-Alvin E. Tanenholtz ArmrneyMichae1 S. Striker ABSTRACT:6-hydroxy-3-keto-A"-steroids of the pregnune or androstane series aremade by fermenting a SB-hydroxy or 3B-acyloxy-5,6-epoxy steroid of thepregnane or androstane series which is saturated in the A-ring withbacteria of the genus Bacillus, Mycobacterium or Arthrobacter or enzymesthereof.

PROCESS FOR MAKING 6-HYDROXY-3-KETO-Al ,4- S'IEROIDS OF THE PREGNANE ANDANDROSTANE SERIES BACKGROUND OF THE INVENTION smoothly proceedingreactions by using specific types of microorganisms identified below.

SUMMARY OF THE INVENTION According to the invention,6-hydroxy-3-keto-A'--steroids of the pregnane and androstane series aremade by fermenting a 3B-hydroxy or 3fl-acyloxy-5,6-epoxy steroid of thepregnane or androstane series which is saturated in the A-ring withbacteria of the genus Bacillus, Mycobacterium or Arthrobacter or enzymesthereof.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Among the above-identifiedgenera of micro-organisms the preferred species are Bacillus lentus,bacillus sphaericus, mycobacterium phlei, Mycobacterium smegmatis orArthrobacter simplex.

lt is essential for the process of the invention that the steroid usedas starting product has a 3Bhydroxyor BB-acyloxygroup and additionallyhas an aor B-oriented 5,6-epoxy ring. ln addition, it may have varioussubstituents which are more or less a matter of choice. For instance,the steroid may have free or functionally modified hydroxyl groups, forinstance in the I 1a, 1 1B, 140:, 15a, 16,- l7-and/or 2l-position. Itmay also have lower alkyl groups, for instance in the 701, 16- and/orl8-position, free or functionally modified keto groups, for in-' stancein the l land/or 17- or 20-position or halogen atoms, preferablychlorine or fluorine, for instance in the lfi-position. The steroid mayalso include additional double bonds, for instance in the 7-, 9(1 1 l4(l and/or lo-position.

The 6-hydroxyl group which is introduced by the process of the inventionmay be aor B-oriented depending on the 5-,6- epoxy ring. 1

The microbiological conversion of the starting products may be carriedout, for instance, in the following manner. By preliminary tests thereare, in the first place, ascertained the optimum fermentation conditionssuch as the selection of a suitable nutritive medium, a suitablesubstrate solvent, the preferable substrate concentration, the technicalconditions such as temperature, airing, pH, stirring, and the optimumtimes for germination and addition of the substrate, as well as the mostfavorable contact time. All these elements are determined analytically,particularly by thinlayer chromatography.

Using the thus-predetermined reaction conditions, the micro-organism isthen subjected to initial cultivation in the nutrient medium bysubmersion under aerobic conditions, is then further multipliedwhereupon the substrate is added in the form of a solution orsuspension. The course of the fermentation is analytically observed ortraced by means of thinlayer chromatography.

Upon completion of the fermentation, the reaction product is extractedfrom the culture broth with a suitable organic solvent, and is isolatedfrom the extract, for instance, by means of evaporation concentrationand conventional purification methods such as recrystallization and/orchromatography.

There are thus obtained 6-hydroxy-3-keto-Asteroids of the pregnane andandrostane series. An example of the products obtained isfia-hydroxy-l-dehydrd testololactone.

Other examples of the products of the invention have, for instance. theformula wherein R is =0 or ,BOH or H, R; is OH or H, R is CH; or Cl-lOH, R is aOH or H, R is a-methyl or H, and R and R together may alsoform an a-epoxy ring in conjunction with the adjoining carbon atoms.

Specific examples of compounds coming under this formula, are, e.g., thefollowing:

60:, l SB-dihydrol 6a-methyll ,4-pregnadiene-3 ,l 1 ,2o-

trione;

611,2 1 -dihydroxyl a-methyll ,4-pregnadiene-3 ,2o-dione;

604,2 l-dihydroxyl ,4-pregnadiene3 ,Zo-dione;

oa-hydroxyl oa-methyl-l ,4-pregnadiene3,2o-dione;

6a, 1 7a-dihydroxyl ,4-pregnadiene-3,2o-dione;

6a,] 13,2 1 -trihydroxyl ,4-pregnadiene-3,2o-dione;

601,1 13,2 l-trihydroxyl 6a-methyll ,4-pregnadiene-3 ,2o-

dione;

6a-hydroxy- 1 6a, l7a-epoxy-1 ,4-pregnadiene-3 ,2o-dione;

dione.

The process of the invention constitutes a technically facile method formaking 6-hydroxy-3-keto-A"-steriods of the pregnane and androstaneseries. These compounds are valuable intermediates for making productshaving hormonal activity.

For instance, the 601,11,8,2l-trihydroxy-loa-methyl-1,4-pregnadiene-3,2o-dione can be converted into the ll/3,2ldihydroxyloa-methyll ,4,6-pregnatriene-3 ,2o-dione, which in turn can be reactedwith hydrogen fluoride and N- bromosuccinimide to form the6a-fluoro-7a-bromo-l 13,21- dihydroxyl 6a-methyl- 1,4-pregnadiene-3,2o-dione. The latter compound may then be subjecteddebromination by hydrogenation and subsequent acid catalystisomerization of the 6B-fluorine substituent. Thus it is possiblewithout any difficulty to form the6a-fluoro-l13,2l-dihydroxy-la-methyll,4-pregnadiene-3,2o-dione(Fluorcortolon").

It is also well known that some of the 6-hydroxy steroids themselveshave valuable pharmaceutical properties. An example is the6B-hydroxy-prednisone (US. Pat. No. 3,058,889), which has anantiinflammatory action.

Making of the Starting Product As has been indicated, the startingproduct is a SB-hydroxyor 3B-acetoxy-epoxy-steroid. These steroids inturn are made from the corresponding 3B-hydroxy or 3B-acyloxy-A-steroids which are subjected to an epoxidation of the A -double bond,for instance by means of peracids.

The following will illustrate the making of the starting product bymeans of a specific example. Sixty g. of3B-hydroxy-2l-acetoxy-l6a-methyl-5- -pregnaene-20-one were dissolved in1.8 l. of dry methylene chloride and reacted with 12 g. sodium acetate(in melted condition), and 40 g. of sodium sulfate (dessicated). At 2C., 36 ml. peroxyacetic acid were then added dropwise while stirringduring a time of 30 minutes. Thereafter, 12 g. of sodium acetate and 14g. of sodium sulfate were again added and 36 ml. of peroxyacetic acidwere again added dropwise during the same period of time as previously.The mass was then subjected to further stirring causing the solution towarm up to room temperature during the course of 2 hours.

After neutralization with 1 liter of a 5% NaHCO solution, whilestirring, separation of the methylene chloride was then effected and thesolution was washed with water, FeSO solution, and then again water, andthen was dried over Na SO and concentrated in vacuo at 40 C. The residuewas subjected to recrystallization from acetic acid ester. There wasthus ob tained 3B-hydroxy-2lacetoxy-5a,6a-epoxy-16a-methylpregnane--one, melting point 167169 C.Yield: about 85 percent.

lf 3-acetates were desired, they could be obtained by epoxidation of theanalogous A"-3-acetates or also by subsequent acetylation of the3B-hydroxy-5oz,6a-epoxy compounds.

The following examples will further illustrate the invention.

EXAMPLE 1 A conical flask was filled with 500 ml. of sterilized aqueousmedium. The medium contained 0.1 percent yeast extract, 0.5 percent cornsteep liquor and 0.05 percent starch sugar.

The mass was adjusted to a pH of 7.0. lt was then inoculated with alyophilized culture of Arthrobacrer simplex and was subjected to shakingfor 48 hours at 30 C. and 145 r.p.m. A 201. fermenter was then chargedwith 14.75 1. of a sterilized nutrient medium of the same compositionand was inoculated with 250 ml. of the above-described bacterialsuspension. The mass was subjected to shaking for 24 hours at 29 C.while airing with 16501. per hour at 220 r.p.m.

0.9 liter of this preliminary fennentation were transferred to a20-liter fermenter, which was charged with 14.1 liter of a sterilizedmedium of the same composition. The principal fermentation mass was thensubjected to the same procedure as employed in the preliminaryfermentation. The pH value during the principal fermentation wasmaintained between 6 and 7. After an initial phase of 6 hours, 3.75 g.of 3B-hydroxy-5 a,6a-epoxy-testololactone in 80 ml. dimethylformamidewere added and subjected to fermentation. The course of the reaction wastraced by thin-layer chromatography of methylisobutylketone extracts ofindividual specimens. The complete con version of the starting materialwas effected after about 34 hours.

The culture was then stirred with methylisobutylketone and the extractwas concentrated at a maximum bath temperature of 40 C. in a vacuumuntil dry. The residue was washed with a small amount of cold hexane andthere were thus obtained, after drying, 3.2 g. of6a-hydroxy-1-dehydrotestololactone, which after recrystallization frommethanol had a melting point between 297 and 299 C. e =15,820.

EXAMPLE 2 The same process was used as in example 1, but a lyophilizedculture was employed of Bacillus lentus instead of Arthrobacter simplex.Furthermore, the initial culture medium was an aqueous medium which hadbeen adjusted to a pH of 7 and contained 1.5 percent of peptone, 1.2percent of comsteep liquor, 0.2 percent MgSO, (instead of the yeastextractcomsteep-glucose medium). The medium for the preliminary and mainfermentation otherwise was the same as in example 1.

The reaction was complete after a contact time of 28 hours. There werewas thus obtained 3.1 g. of crude product which, after recrystallizationfrom methanol, furnished 2.2 g. 6ahydroxy- 1 -dehydrotestololactone;m.p. 293295 C.

The compound was the same as the product obtained in example 1, asascertained by a comparison of the spectra and the mixture meltingpoints.

EXAMPLE 3 1n the same manner as in example 2, there were dissolved 3.75g. of 3,8-hydroxy-Sa,6a6a-epoxy-androstane-17-one in 80 ml.dimethylformamide which were then fermented with Bacillus lentus. Thereaction was complete after 28 hours. The crude product obtained (3.4g.) was recrystallized from methanol. There were thus obtained 2.4 g. of6a-hydroxy-l,4 androstadiene-3,17-dione; m.p. 255258 C. e =15,100.

EXAMPLE 4 The same conditions were used as in example 2. 2.7 g. of3B,15B-dihydroxy-5a,6a-epoxy-16-methyl-pregnane-1 1,20- dione dissolvedin 30 ml. dimethylformamide were fermented with Bacillus lentus. After acontact time of 27 hours, the reaction was complete. By furtherprocessing, as in the previous examples, there were obtained 2.2 g. of6a,15,8-dihydroxy- 16a-methyl-1,4-pregnadiene-3,1 1,20-trione which,after recrystallization from acetic acid ester, has a melting point of263-267 C. e =14,100.

A specimen of this product was then subjected to acetylation inpyridine/acetic acid anhydride at room temperature. There was thusobtained the corresponding 6a,15B-diacetate. The melting point of thisproduct was 202 to 204 C; e =14,800.

EXAMPLE 5 Following again the procedure of example 2, 3.75 of 3B-hydroxy-2 1-acetoxy-5a,6a-epoxy-16a-methyl-pregnane-20- one werefermented for 27 hours with Bacillus lentus and then subjected tofurther processing. By column chromatography and recrystallization fromacetone/isopropylether, there was obtained 601,2 l-dihydroxy- 16a-methyl- 1 ,4-pregnadiene-3 ,20- dione; m.p. 207209 C.; e =l 5,400.

EXAMPLE 6 Following the procedure of example 2, 3.0 g. of3,8-hydroxy-21-acetoxy-5a,6u-epoxy-pregnane-20-one were fermented for 30hours in 12 liters of Bacillus lemus culture, and then subjected tofurther processing. By column chromatography, and recrystallization fromacetone/isopropyl-ether, there were obtained 601,21,-dihydroxy-l,4-pregnadiene-3,20-dione; m.p. 187Al89 C.; e =15,700.

EXAMPLE 7 Following the procedure of example 2, 3.75 g. of 3B-hydroxy-5a,6a-epoxy-16-methyl-pregnane-20-one were fermented for 28hours with Bacillus lentus and then subjected to further processingafter column chromatography and recrystallization from acetone/hexane,there was obtained hydroxy-l6a-methyl-1,4-pregnadiene-3,20-dione; m .p.178-180 C.; e ,=l4, 100.

EXAMPLE 8 EXAMPLE 9 Following the procedure of example 1, 1.87 g. of38,113- 21-trihydroxy-5a,6a-epoxy-pregnane-20-one were fermented in 15liters of culture broth of Arthrobacter simplex and subjected tofermentation for 20 hours and then subjected to further processing. Bycolumn chromatography of the crude product and recrystallization fromacetic acid ester/isopropylether, there were obtained 611,113,21-trihydroxyl ,4-pregnadiene-3,20-dione. m.p. 2 l O/2l 1 212 C.; e=l4,2OO.

EXAMPLE 10 Following the procedure of example 1, 1.87 g. of 33,1 15,21-trihydroxy-5a,6a-epoxy- 1 6a-methyl-pregnane-20-one were fermented in15 liters of a culture broth of Arlhrobacter simplex for 27 hours andthen subjected to further processing. By

EXAMPLE 1 1 Following the procedure of example 1, 2 g. of 3B-hydroxy-518,6/3-epoxy-testololactone were dissolved in 50 ml. dimethylfonnarnide,were fermented in 8 l. of a culture broth for a period of 56 hours withArlhrobacter simplex. By further treatment as above described, an oilycrude product (1.5 g.) was obtained and separated by columnchromatography on silica gel. After recrystallization from acetic acidester/isopropyl ether, there was obtained6B-hydroxy-l-dehydro-testololactone; m.p. 254-258 C.; 16,000.

EXAMPLE 12 Following the procedure of example 2, 3.75 g. of 3B-hydroxy-5a,6a, 1 6a, 1 7a-diepoxy-pregnane-20-one were fermented in 15liter of a culture broth of Bacillus lentus. After 29 hours, thereaction was complete. The crude product (2.93 g.) which was obtained bythe treatment as described was then recrystallized from acetic acidester and furnishedoar-hydroxy-l6a,17a-epoxy-1,4-pregnadiene-3,20-dione; m.p. 227230 C.; e=l4,500.

EXAMPLE 13 Following the procedure of example 2, 3.75 g. of 3B-hydroxy-2 l acctoxy-5a,6a, 1 6a, 1 7a-diepoxy-pregnane-20one werefermented with Bacillus lentus. After 26 hours, the reaction wascomplete. The crude product (3.3 g.) which was obtained by the treatmentas described was recrystallized from acetic acid ester-isopropyletherand furnished 6a,2ldihydroxy- 1 6a- 1 7a-epoxyl ,4-pregnadiene-3,20dione; m.p. 5560 C.; e =l 3,900.

EXAMPLE 14 A lyophilized culture of Bacillus sphaericus ATCC 7055 wasintroduced into a solution of 500 ml. of a sterile, aqueous medium whichcontained 0.1% peptone, 0.2% comsteep liquor, 0.5% glucose, and 0.5%yeast extract, and which was adjusted to a pH of 7. The mass wassubjected to shaking for 48 hours at 30 C. 15 ml. of the bacterialsuspension were then transferred to a 2-liter chemical flask which wasfilled with 500 ml. of the same medium. After 24 hours, 50 ml. of theculture broth, each, were transferred into four conical flasks which hadbeen filled, each, with 500 ml. of the same medium. After shaking for 6hours, there were added to each flask 100 mg. of 3,8-hydroxy-5a,6a-epoxy-androstanel 7-one dissolved in 5 ml.dimethylforrnamide. The mass was then shaken for another 42 hours at 30C. Thereafter, the fermentation broth was shaken with methylisobutylketone. An adequate amount of the extract was then testedagainst a comparison amount of a standard which was6a-hydroxy-l,4androstadiene-3,l7- dione. This test was carried out onsilica gel plates by thinlayer chromatography; Rf 0.37 (benzene/aceticacid ester 1:4) The extract which contained about 70 -80 percent of theconverted product was then further processed in the described manner bycolumn chromatography and the separated crude product was recrystallizedfrom acetone/isopropylether. There were thus obtained 275 mg. ofa-hydroxy-l ,4-androstadiene-3, l 7-dione; m.p. 257259 C.

EXAMPLE 15 A lyophilized culture of Mycobacterium phlei was subjected toinitial cultivation and fermentation. The substrate was again 400 mg. of3B-hydroxy-5a,6a-epoxy-androstanel 7-one.

The fennentation time was 42 hours. After thin-layer chromatography,there were again obtained 17 -80 percent of 6ahydroxyl,4-androstandiene-3 ,17-dione (Rf 0.37

benzene/acetic acid ester 1:4). After further processing of the mass asabove there were obtained 260 mg. of 6a-hydroxyl ,4-androstadiene-3,17-dione; m.p. 255-259 C.

EXAMPLE 16 A lyophilized culture of Mycabacterium .rmegmatis ATCC 14468was subjected to initial cultivation as in example 14. The substrate wasagain 400 mg. of 3,8hydroxy-5a,6a-epoxyandrostane-17-one. Thefermentation time was 64 hours. After thin-layer chromatography, therewere obtained about 50 percent ofGa-hydroxy-1,4-androstadiene-3,l7-dione. After further treatment asdescribed above, there were ob tained 180 mg. of6a-hydroxy-l,4-androstadiene-3,l7-dione; m.p. 254-257 C.

Without further analysis, the foregoing will so fully reveal the gist ofthe present invention that others can by applying current knowledgereadily adapt it for various applications without omitting featuresthat, from the standpoint of prior art, fairly constitute essentialcharacteristics of the generic or specific aspects of this inventionand, therefore, such adaptations should and are intended to becomprehended within the meaning and range of equivalence of thefollowing claims.

What is claimed as new and desired to be protected by Letters Patent isset forth in the appended 1. The process of making6-hydroxy-3-keto-A--steroids of the pregnane or androstane seriescomprising fermenting a 3B-hydroxyor 3B-acyloxy-5,6-epoxy steroid of thepregnane or androstane series which is saturated in the A-ring withbacteria of the genus Bacillus, Mycobacterium or Arthrabacter or enzymesthereof.

2. The process of claim 1, wherein the bacteria belong to the speciesBacillus lentus, Bacillus sphaericus, Mycobaclerium phlei, Mycobacleriumsmegmatis or Arthrobaclerium simplex.

3. The process of claim 1 wherein said steroid subjected to fermentationis 3B-hydroxy-5a,a-epoxy-testololactone, said bacteria is Arthmbactersimplex and there is recovered 6ahydroxy-1-dehydro-testololactone.

4. The process of claim 1 wherein said steroid subjected to fermentationis 3B-hydroxy-5a,6a-epoxy-testololactone, said bacteria is Bacilluslentus and there is recovered 6a-hydroxy-l -dehydro-testololactone.

5. The process of claim 1 wherein said steroid subjected to fermentationis 3/3-hydroxy-5a,6a-epoxy-androstane-l7-one, said bacillus is Bacillus[cams and there is recovered 6ahydroxy-l ,4-androstadiene-3 ,l 7 -dione.

6. The process of claim 1 wherein said steroid is 313,155-dihydroxy-5a,6a-epoxyl 6a-methyl-pregnane-l l ,ZO-dione, said bacillusis Bacillus lentus and there is recovered 601,151?-dihydroxy-16a-methyl-l,4-pregnadiene-3,l 1,20-trione.

7. The process of claim 1 wherein said steroid subjected to fermentationis 3a-hydroxy-2 l-acetoxy-5a,6a-epoxy- 1 6amethyl-pregnane-ZO-one, saidbacillus is Bacillus lentus and there is recovered6a,2l-dihydroxy-la-methyl-l,4- pregnadiene-3,20-dione.

8. The process of claim 1 wherein said steroid subjected to fermentationis 3,8-hydroxy-2 l -acetoxy-5a,6a-epoxypregnane-ZO-one, said bacillus isBacillus lentus and there is recovered 601,2 l-dihydroxy- 1,4-pregnadiene-3,20-dione.

9. The process of claim 1 wherein said steroid subjected to fermentationis 3B-hydroxy-5a,6a-epoxyl 6-methylpregnane-ZO-one, said bacillus isBacillus lenms and there is recovered 6a-hydroxyl oa-methyl- 1,4-pregnadiene-3 ,20- dione.

10. The process of claim 1 wherein said steroid subjected tofermentation is 3B,17a-dihydroxy-Sa,6a-epoxy-pregnane-20- one, saidbacillus is Bacillus lentus and there is recovered6a,l7a-dihydoxy-1,4-pregnadiene 3,20-dione.

1 1. The process of claim 1 wherein said steroid subjected tofermentation is 33, 1 13,2 1 -trihydroxy-5a,-6a-epoxypregnane, saidbacteria is Arthrobacter simplexand there is recovered 601,1 13,2l-trihydroxy-l ,4pregnadiene-3,20-dione.

12. The process of claim 1 wherein said steroid subjected tofermentation is 33,1 13,2 l-trihydoroxy-a6a-epoxy-16amethyl-pregnane,said bacteria is Arthrobacrer simplex and there is recovered 6a,l15,2l-trihydroxy-l6a-methyl-1,4- pregnadiene-3,20-dione.

13. The process of claim 1 wherein said steroid subjected tofermentation is 3/3-hydroxy-5B,6/3-epoxy-testololactone, said bacteriais Arthrobacrer simplex and there is recovered 6;?- hydroxyl-dehydro-testololactone.

14. The process of claim 1 wherein said steroid subjected tofermentation is 3B-hydroxy-5a,6a, 1 6a, 1 7a-diepoxypregnane-ZO-one,said bacteria is Bacillus Ientus and there is recovered 6a-hydroxy-16a,l7a-epoxy-l ,4-pregnadiene-3,20- dione.

15. The process of claim 1 wherein said steroid subjected tofermentation is 3B-hydroxy-2 l-acetoxy-5a,6a- 1 6a, l7adiepoxy-pregnane-ZO-one, said bacteria is Bacillus lenrus and there isrecovered 6a,2l-dihydroxy-,l6a,l7a-epoxy-1,4- pregnadiene-3,20-dione.

16. The process of claim 1 wherein said steroid subjected tofermentation is 3B-hydroxy-5a,6a-epoxy-androstane-l7-one, said bacteriais Bacillus sphaericus and there is recovered 6ahydroxyl,4-androstiene-3 l 7-dione.

17. The process of claim 1 wherein said steroid subjected tofermentation is 3B-hydroxy-5a,6a-epoxy-androstanel 7 one, said bacteriais Mycabacterium phlei and there is recovered6a-hydroxy-1,4-androstadiene-3 ,7-dione.

18. The process of claim 1 wherein said steroid subjected tofennentation is 3B-hydroxy-5a,6a-epoxy said bacteria is Mycobacteriumsmegmalic and there is recovered 6a-hydroxyl ,4-androstadiene-3, l7-dione.

l I. t I i

2. The process of claim 1, wherein the bacteria belong to the speciesBacillus lentus, Bacillus sphaericus, Mycobacterium phlei, Mycobacteriumsmegmatis or Arthrobacterium simplex.
 3. The process of claim 1 whereinsaid steroid subjected to fermentation is 3 Beta -hydroxy-5 Alpha ,6Alpha -epoxy-testololactone, said bacteria is Arthrobacter simplex andthere is recovered 6 Alpha -hydroxy-1-dehydro-testololactone.
 4. Theprocess of claim 1 wherein said steroid subjected to fermentation is 3Beta -hydroxy-5 Alpha ,6 Alpha -epoxy-testololactone, said bacteria isBacillUs lentus and there is recovered 6 Alpha-hydroxy-1-dehydro-testololactone.
 5. The process of claim 1 whereinsaid steroid subjected to fermentation is 3 Beta -hydroxy-5 Alpha ,6Alpha -epoxy-androstane-17-one, said bacillus is Bacillus lentus andthere is recovered 6 Alpha -hydroxy-1,4-androstadiene-3,17-dione.
 6. Theprocess of claim 1 wherein said steroid is 3 Beta ,15 Beta -dihydroxy-5Alpha ,6 Alpha -epoxy-16 Alpha -methyl-pregnane-11,20-dione, saidbacillus is Bacillus lentus and there is recovered 6 Alpha ,15 Beta-dihydroxy-16 Alpha -methyl-1,4-pregnadiene-3,11,20-trione.
 7. Theprocess of claim 1 wherein said steroid subjected to fermentation is 3Alpha -hydroxy-21-acetoxy-5 Alpha ,6 Alpha -epoxy-16 Alpha-methyl-pregnane-20-one, said bacillus is Bacillus lentus and there isrecovered 6 Alpha ,21-dihydroxy-16 Alpha-methyl-1,4-pregnadiene-3,20-dione.
 8. The process of claim 1 whereinsaid steroid subjected to fermentation is 3 Beta -hydroxy-21-acetoxy-5Alpha ,6 Alpha -epoxy-pregnane-20-one, said bacillus is Bacillus lentusand there is recovered 6 Alpha ,21-dihydroxy-1,4-pregnadiene-3,20-dione.9. The process of claim 1 wherein said steroid subjected to fermentationis 3 Beta -hydroxy-5 Alpha ,6 Alpha -epoxy-16-methyl-pregnane-20-one,said bacillus is Bacillus lentus and there is recovered 6 Alpha-hydroxy-16 Alpha -methyl-1,4-pregnadiene-3,20-dione.
 10. The process ofclaim 1 wherein said steroid subjected to fermentation is 3 Beta ,17Alpha -dihydroxy-5 Alpha ,6 Alpha -epoxy-pregnane-20-one, said bacillusis Bacillus lentus and there is recovered 6 Alpha ,17 Alpha-dihydoxy-1,4-pregnadiene 3,20-dione.
 11. The process of claim 1 whereinsaid steroid subjected to fermentation is 3 Beta ,11 Beta,21-trihydroxy-5 Alpha ,-6 Alpha -epoxy-pregnane, said bacteria isArthrobacter simplex and there is recovered 6 Alpha ,11 Beta,21-trihydroxy-1, 4pregnadiene-3,20-dione.
 12. The process of claim 1wherein said steroid subjected to fermentation is 3 Beta ,11 Beta,21-trihydoroxy-5 Alpha 6 Alpha -epoxy-16 Alpha -methyl-pregnane, saidbacteria is Arthrobacter simplex and there is recovered 6 Alpha ,11 Beta, 21-trihydroxy-16 Alpha -methyl-1,4-pregnadiene-3,20-dione.
 13. Theprocess of claim 1 wherein said steroid subjected to fermentation is 3Beta -hydroxy-5 Beta ,6 Beta -epoxy-testololactone, said bacteria isArthrobacter simplex and there is recovered 6 Beta-hydroxy-1-dehydro-testololactone.
 14. The process of claim 1 whereinsaid steroid subjected to fermentation is 3 Beta -hydroxy-5 Alpha ,6Alpha ,16 Alpha ,17 Alpha -diepoxy-pregnane-20-one, said bacteria isBacillus lentus and there is recovered 6 Alpha -hydroxy-16 Alpha ,17Alpha -epoxy-1,4-pregnadiene-3,20-dione.
 15. The process of claim 1wherein said steroid subjected to fermentation is 3 Beta-hydroxy-21-acetoxy-5 Alpha ,6 Alpha -16 Alpha ,17 Alpha-diepoxy-pregnane-20-one, said bacteria is Bacillus lentus and there isrecovered 6 Alpha ,21-dihydroxy-, 16 Alpha ,17 Alpha-epoxy-1,4-pregnadiene-3,20-dione.
 16. The process of claim 1 whereinsaid steroid subjected to fermentation is 3 Beta -hydroxy-5 Alpha ,6Alpha -epoxy-androstane-17-one, said bacteria is Bacillus sphaericus andthere is recovered 6 Alpha -hydroxy-1,4-androstadiene-3,17-dione. 17.The process of claim 1 wherein said steroid subjected to fermentation is3 Beta -hydroxy-5 Alpha ,6 Alpha -epoxy-androstane-17-one, said bacteriais Mycobacterium phlei and there is recovered 6 Alpha-hydroxy-1,4-androstadiene-3,7-dione.
 18. The process of claim 1 whereinSaid steroid subjected to fermentation is 3 Beta -hydroxy-5 Alpha ,6Alpha -epoxy said bacteria is Mycobacterium smegmatic and there isrecovered 6 Alpha -hydroxy-1,4-androstadiene-3,17-dione.